Optimization of Milk Sample Cleanup Using Response Surface Methodology

  • 129 Accesses


Protein precipitation is a fast and simple methodology for sample preparation in milk, performed by addition of an organic solvent compatible with the analytical system. Therefore, the aim of this study is to compare four organic solvents efficiency: (i) acetonitrile, (ii) acetone, (iii) ethanol, and (iv) isopropanol for deproteinization. Acetonitrile was the most efficient at freezer temperature, while at room temperature the most efficient solvent was acetone. In order to optimize precipitation conditions, two central composite designs (CCDs) associated with response surface methodology (RSM) were performed using acetonitrile as precipitant. ANOVA was applied to the experimental designs and data were adjusted to quadratic model with high order of significance, suitable adjustment of the experimental data, with Adjusted-R2 > 0.98, and adequacy precision desirable. For models’ optimization and validation, the desirability function was performed, and under the optimized conditions, which were obtained with a total desirability value of 1.000, the experimental values are statistically equal to predicted values for protein. The volume of precipitant and the ultrasound time were significant on the response, whereas the vortex time was a non-signifcant factor. The RSM was applied efficiently in the optimization process allowing the simultaneous evaluation of the variables on the response.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Behera SK, Meena H, Chakraborty S, Meikap B (2018) Application of response surface methodology (RSM) for optimization of leaching parameters for ash reduction from low-grade coal. Int J Mining Sci Technol 28 (4):621–629.

  2. Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76(5):965–977.

  3. Candioti LV, Zan MMD, Cámara MS, Goicoechea HC (2014) Experimental design and multiple response optimization. using the desirability function in analytical methods development. Talanta 124:123–138.

  4. Derringer G, Suich R (1980) Simultaneous optimization of several response variables. J Qual Technol 12 (4):214–219.

  5. Fakheri F, Moghaddas J, Zadghaffari R, Moghaddas Y (2012) Application of central composite rotatable design for mixing time analysis in mechanically agitated vessels. Chem Eng Technol 35(2):353–361.

  6. Fic E, Kedracka-Krok S, Jankowska U, Pirog A, Dziedzicka-Wasylewska M (2010) Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis. Electrophoresis 31 (21):3573–3579.

  7. Freitas A, Barbosa J, Ramos F (2013) Development and validation of a multi-residue and multiclass ultra-high-pressure liquid chromatography-tandem mass spectrometry screening of antibiotics in milk. Int Dairy J 33(1):38–43.

  8. Fritz R, Ruth W, Kragl U (2009) Assessment of acetone as an alternative to acetonitrile in peptide analysis by liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 23(14):2139–2145.

  9. Garballo-Rubio A, Soto-Chinchilla J, Moreno A, Zafra-Gómez A (2018) Determination of residual lactose in lactose-free cow milk by hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry. J Food Compos Anal 66:39–45.

  10. Heaton J, Jones MD, Legido-Quigley C, Plumb RS, Smith NW (2011) Systematic evaluation of acetone and acetonitrile for use in hydrophilic interaction liquid chromatography coupled with electrospray ionization mass spectrometry of basic small molecules. Rapid Commun Mass Spectrom 25 (24):3666–3674.

  11. Jambrak AR, Mason TJ, Lelas V, Herceg Z, Herceg IL (2008) Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions. J Food Eng 86(2):281–287.

  12. Kamizake NK, Gonçalves M M, Zaia CT, Zaia DA (2003) Determination of total proteins in cow milk powder samples: a comparative study between the kjeldahl method and spectrophotometric methods. J Food Compos Anal 16(4):507–516.

  13. Kim HJ, Bae IK, Jeong MH, Park HJ, Jung JS, Kim JE (2015) A new HPLC-ELSD method for simultaneous determination of n-acetylglucosamine and n-acetylgalactosamine in dairy foods. Int J Anal Chem 2015:1–8.

  14. McMaster M (2005) Appendix b: solvents and volatile buffers for LC/MS. In: LC/MS. Wiley, pp 139–142.

  15. McPherson A (2004) Introduction to protein crystallization. Methods 34(3):254–265.

  16. Neto BB, Scarminio IS, Bruns RE (2010) Como Fazer Experimentos: Pesquisa e Desenvolvimento na Ciência e na Indústria. Bookman Editora

  17. Neumann C, Harris DM, Rogers LM (2002) Contribution of animal source foods in improving diet quality and function in children in the developing world. Nutr Res 22(1-2):193–220.

  18. Nollet LM (2009) Handbook of dairy foods analysis. CRC Press.

  19. O’sullivan J, Murray B, Flynn C, Norton I (2016) The effect of ultrasound treatment on the structural, physical and emulsifying properties of animal and vegetable proteins. Food hydrocolloids 53:141–154

  20. Palombini SV, Claus T, Maruyama SA, Carbonera F, Montanher PF, Visentainer JV, Gomes STM, Matsushita M (2016) Optimization of a new methodology for determination of total phenolic content in rice employing fast blue BB and QUENCHER procedure. Journal of the Brazilian Chemical Society.

  21. Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R (2003) Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography–tandem mass spectrometry. J Chromatogr B 785(2):263–275.

  22. Privalov PL (1990) Cold denaturation of protein. Critical reviews in biochemistry and molecular biology 25 (4):281–306

  23. R Development Core Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.

  24. Raza N, Kim KH (2018) Quantification techniques for important environmental contaminants in milk and dairy products. TrAC Trends Anal Chem 98:79–94.

  25. Riekkola ML (2009) Editorial on “effect of eluent on the ionization process in liquid chromatography–mass spectrometry” by r. kostiainen and t. kauppila. J Chromatograp 1216(4):684.

  26. Silva E, Pompeu D, Larondelle Y, Rogez H (2007) Optimisation of the adsorption of polyphenols from inga edulis leaves on macroporous resins using an experimental design methodology. Sep Purif Technol 53(3):274–280.

  27. Souverain S (2004) Protein precipitation for the analysis of a drug cocktail in plasma by LC-ESI-MS. Journal of Pharmaceutical and Biomedical Analysis.

  28. Waterborg JH (2009) The Lowry method for protein quantitation. In: Protein protocols handbook. The Humana Press, pp 7–10.

  29. Xian Y, Dong H, Wu Y, Guo X, Hou X, Wang B (2016) QuEChERS-based purification method coupled to ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to determine six quaternary ammonium compounds (QACs) in dairy products. Food Chem 212:96–103.

  30. Yanjun S, Jianhang C, Shuwen Z, Hongjuan L, Jing L, Lu L, Uluko H, Yanling S, Wenming C, Wupeng G, Jiaping L (2014) Effect of power ultrasound pre-treatment on the physical and functional properties of reconstituted milk protein concentrate. J Food Eng 124:11–18.

  31. Zaia D, Verri W, Zaia C (2000) Determination of total proteins in several tissues of rat: a comparative study among spectrophotometric methods. Microchem J 64(3):235–239.

Download references

Author information

Correspondence to Lucas Ulisses Rovigatti Chiavelli.

Ethics declarations

Conflict of interest

Lucas Ulisses Rovigatti Chiavelli declares that he has no conflict of interest. Antonio Cesar Godoy declares that he has no conflict of interest. Roberta da Silveira declares that he has no conflict of interest. Patricia Daniele Silva Santos declares that she has no conflict of interest. Tiago A. M. Lopes declares that he has no conflict of interest. Oscar Oliveira Santos declares that he has no conflict of interest. Jesuí Vergílio Visentainer declares that he has no conflict of interest.

Additional information

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chiavelli, L.U.R., Godoy, A.C., Silveira, R.d. et al. Optimization of Milk Sample Cleanup Using Response Surface Methodology. Food Anal. Methods 13, 166–175 (2020) doi:10.1007/s12161-019-01567-8

Download citation


  • Milk
  • Sample preparation
  • Protein precipitation
  • Precipitant solvent
  • Central composite design